All bacteria can be separated into one or two classes, either gram-positive or gram-negative, based on their gram-stain reaction. The gram reaction is, therefore, a key test in the identification of bacteria. In addition, because of general structural and chemical differences between gram-positive and gram-negative bacteria, a different spectrum of antibiotics is used to treat infections caused by one class than is used to treat infections caused by the other class. Knowledge of the gram reaction of an infecting organism is, therefore, important for determination of appropriate treatment.
Currently, the gram stain reaction is a four-step staining procedure performed on a glass slide containing heat-fixed biological specimens. This procedure is both time consuming and labor intensive. Bartholomew (The Gram Stain, Bact. Rev., 16, pp. 1-29, 1952) has written a comprehensive article describing this procedure. It is well known that gram staining can give varying results and is highly dependent upon precise timing and meticulous technique. Furthermore, the procedure is difficult to automate.
An improved differentiation method using anionic surfactants is described in U.S. Pat. No. 4,525,453 (issued Jun. 25, 1985). According to that reference, anionic surfactants selectively inhibit the ability of gram-positive organisms to reduce certain reducible compounds. One of the surfactants taught as preferred in that reference is marketed under the trademark TERGITOL 7 (Sigma Chemical Co., St. Louis, MO.)
It has been found, however, that the anionic surfactants taught in the art do not effectively differentiate organisms with a wide range of reducible compounds. For example, TERGITOL 7 is useful with tetrazolium salts, but is not useful with certain reducible intramolecular nucleophilic displacement compounds (identified as RIND compounds herein). In other words, no clear gram separation was observed using a combination of the anionic surfactants and the RIND compounds.
It would be desirable to have a differentiation method which is not only rapid and simple, but also would be useful with a broad range of reducible materials.